Hapten-carrier conjugates for treating and preventing nicotine addiction

ABSTRACT

Novel hapten-carrier conjugates are capable of inducing the production of antibodies, in vivo, that specifically bind to nicotine. These conjugates comprise a nicotine hapten conjugated to an immunogenic carrier protein. The novel conjugates preserve the chirality of nicotine in its native (S)-(−) state, and have good stability properties. The conjugates are useful in formulating vaccines for active immunization, that are used to prevent and treat nicotine addiction. The antibodies raised in response to the nicotine hapten-carrier conjugate are used for passive immunization. These antibodies are administered for prevention and treatment of nicotine addiction.

FIELD OF THE INVENTION

[0001] The present invention relates to treatment and prevention ofnicotine addiction. In particular, the invention relates to novelhapten-carrier conjugates which are capable of inducing the productionof antibodies. Such antibodies are capable of specifically binding tonicotine. Furthermore, the present invention envisages preventing ortreating nicotine addiction by administering a nicotine-carrierconjugate in a pharmaceutically-acceptable formulation. The presentinvention also contemplates using the antibodies raised in response tothe hapten-carrier conjugate for the prevention and treatment ofnicotine addiction.

BACKGROUND OF THE INVENTION

[0002] Smoking of cigarettes, cigars, and pipes is a prevalent problemin the United States and worldwide. Smoking tobacco and smokelesstobacco are rich in nicotine, which is a known addictive substance.Nicotine is an alkaloid derived from the tobacco plant that isresponsible for smoking's psychoactive and addictive effects. Nicotineis formed of two rings linked together by a single bond: an aromaticsix-membered ring (pyridine) and an aliphatic five-membered ring(pyrrolidine). The pyrrolidine is N-methylated and linked through itscarbon-2 to the carbon-3 of pyridine. Thus, the carbon-2 is chiral, andthere is virtually free rotation around the single bond linking the tworings. It has been established that the absolute configuration ofcarbon-2 is S. Thus, the natural configuration of nicotine is(S)-(−)-nicotine.

[0003] Nicotine use is widespread due to the easy availability ofcigarettes, cigars, pipes and smokeless tobacco. According to the U.S.Department of Health and Human Services, cigarette smoking is the singleleading cause of preventable death in the United States. See alsoMcGinnis et al., J. Am. Med. Assoc., 270, 2207-2211 (1993). Exposure tosecond hand smoke also has been reported to have serious detrimentalhealth effects, including exacerbation of asthma.

[0004] Even though the addictive nature of nicotine is well known,cigarette smoking is prevalent. Peak levels of nicotine in the blood,about 25 to 50 nanograms /ml, are achieved within 10-15 minutes ofsmoking a cigarette. In humans, smoking a cigarette results in arterialnicotine concentrations being 10-fold higher than venous nicotineconcentrations because nicotine is rapidly delivered from the lungs tothe heart (see Henningfield (1993) Drug Alcohol Depend. 33:23-29). Thisresults in a rapid delivery of high arterial concentrations of nicotineto the brain. Once nicotine crosses the blood-brain barrier, evidencesuggests that it binds to cholinergic receptors, which are normallyactivated by the neurotransmitter acetylcholine, which is involved inrespiration, maintenance of heart rate, memory, alertness and musclemovement. When nicotine binds to these receptors, it can affect normalbrain function, by triggering the release of other neurotransmitters,such as dopamine. Dopamine is found in the brain in regions involved inemotion, motivation, and feelings of pleasure. It is the release ofneurotransmitters, especially dopamine, that is responsible for thetobacco user's addiction to nicotine or other intake of nicotine.

[0005] Due to the significant adverse effects of smoking on health,smokers often try to quit. However, the addictive nature of nicotine andthe availability of cigarettes add to the continued dependence onnicotine and high failure rate of those who try to quit. Withdrawalsymptoms are unpleasant, and are relieved by smoking.

[0006] Many therapies for nicotine addiction have been developed, butare largely ineffective. The two most popular therapies remain thenicotine transdermal patch and nicotine incorporated into chewing gum.These therapies, termed “nicotine replacement therapies” (NRT), replacethe amount of nicotine which the user previously received from smokingand act to wean the user off nicotine. However, certain drawbacks areseen with this type of therapy. Particularly, there is low penetrationof nicotine into the bloodstream and therefore an increased desire tosmoke. Problems such as mouth irritation, jaw soreness, nausea, havebeen associated with use of nicotine chewing gum. Problems such as skinirritations, sleep disturbance, and nervousness have been associatedwith use of nicotine transdermal patches.

[0007] Therefore, an alternative methodology for treating nicotineaddiction is needed. The literature recognizes this need and there havebeen several attempts to provide a methodology for treating nicotineaddiction. One of the methods involves the administration of antibodieswhich have been raised in response to nicotine. However, low molecularweight substances, called haptens, are known to be unable to trigger animmune response in host animals. Nicotine is no exception, and as asmall molecule it is not immunogenic. To elicit an antibody response toa hapten, it typically is covalently bound to a carrier protein, and thecomplex will elicit the production of antibodies, that recognize thehapten.

[0008] For example, cotinine 4′-carboxylic acid, when bound covalentlyto keyhole limpet hemocyanin (KLH) was used to generate antibodies tothe nicotine metabolite cotinine. Those antibodies were used todetermine the presence of cotinine in physiological fluids. See Bjerkeet al. J. Immunol. Methods, 96, 239-246 (1987).

[0009] Other nicotine antibodies were prepared by Castro et al., (Eur.J. Biochem., 104, 331-340 (1980)). Castro et al. prepared nicotinehaptens, conjugated to bovine serum albumin (BSA), with the carrierprotein conjugated via a linker at the 6-position of nicotine. Castro etal. prepared additional nicotine conjugates of BSA which were injectedinto mammals to raise antibodies. In another publication, Castro et al.in Biochem. Biophys. Res. Commun. 67, 583-589 (1975) disclose twonicotine albumin conjugates: N-succinyl-6-amino-(±)-nicotine-BSA and6-(σ-aminocapramido)-(±)-nicotine-BSA. In this 1975 publication, Castroet al. also used antibodies to nicotine carrier conjugate,6-(σ-aminocapramido)-(±)-nicotine-BSA, to determine the levels ofnicotine in blood and urine, see Res. Commun Chem. Path. Pharm. 51,393-404 (1986).

[0010] Swain et al. (WO 98/14216) disclose nicotine carrier conjugateswherein the hapten is conjugated at the 1, 2, 4, 5, 6, or 1′ position ofthe nicotine. Hieda et al. have shown that animals immunized with6-(carboxymethylureido)-(±)-nicotine, which was linked to keyhole limpethemocyanin, produced antibodies specific to nicotine. J. Pharm. andExper. Thera. 283, 1076-1081 (1997). Langone et al. prepared the haptenderivative, O-succinyl-3′-hydroxymethyl-nicotine, see Biochemistry, 12,5025-5030, and used the antibodies to this hapten carrier conjugate inradioimmunoassays. See Methods in Enzymology, 84, 628-635 (1982). Theconjugate produced by Langone is susceptible to hydrolysis.Additionally, Abad et al. in Anal. Chem., 65, 3227-3231 (1993) describeconjugating 3′-(hydroxymethyl) nicotine hemisuccinate to bovine serumalbumin to produce antibodies to nicotine in order to be able to measurenicotine content in smoke condensate of cigarettes in an ELISA assay.

[0011] Therefore, the prior art does not teach a stable nicotine-carrierconjugate that preserves the chiral nature of the nicotine hapten, andthat links the hapten to the carrier in a way that conserves the natureof the nicotine epitope(s). Moreover, the art does not teach or suggestmethods of preventing and treating nicotine addiction by using suchconjugates. Seeman in Heterocycles, 22, 165-193, (1984) disclosesresults of a study of the conformational analysis and chemicalreactivity of nicotine.

SUMMARY OF THE INVENTION

[0012] In response to the demand for a more effective methodology fortreating nicotine addiction, it is one object of the present inventionto provide novel nicotine-carrier conjugates that are stable, comprisenicotine in its natural (S)-(−) formation, and employ a nicotine-carrierlinkage that preserves the nature of the nicotine epitope(s), and therelative orientation of the two rings of the nicotine molecule. Bothrings of nicotine, and their relative orientation, are believed to beessential for the recognition by antibody of nicotine in solution. Suchconjugates are capable of stimulating the production of antibodies thatare capable of specifically binding to nicotine. Using the inventiveconjugates, the inventors have raised serum nicotine levels, anddecreased brain nicotine levels, in mammals. Additionally, using theconjugates of the invention, the inventors also have preventednicotine-induced changes in blood pressure, and locomotor effects.

[0013] In another object of the present invention is provided a methodof treating nicotine addiction by administering a conjugate of theinvention to a patient addicted to nicotine thereby generateanti-nicotine antibodies in that patient. Thus, when the patient smokes(or uses chewing tobacco), the nicotine from these products will bebound by the anti-nicotine antibodies in the blood, preventing thenicotine from crossing the blood-brain barrier, hence eliminating thenicotine-induced alterations in brain chemistry, which is the source ofnicotine-addiction. In this regard, it is important that thenicotine-carrier conjugate elicit the production of antibodies that willrecognize the native nicotine molecule. As described above, the novelnicotine-carrier conjugates of the invention preserve the chirality andthe epitope(s) of naturally-occurring nicotine.

[0014] The inventors do not intend to be bound by any particular theoryas to how the nicotine conjugates, and the antibodies produced inresponse to such conjugates, inhibit the effects of nicotine ingested bymammals. In addition to preventing nicotine from crossing the bloodbrain barrier, the antibodies also may prevent nicotine from binding toother receptors in the peripheral nervous system by simple stericblockage.

[0015] These objects can be achieved by providing a hapten-carrierconjugate of formula (I):

[0016] wherein m is 1 to 2500, n is 0 to 12, y is 1 to 12, X is selectedfrom the group consisting of NH—CO, CO—NH, CO—NH—NH, NH—NH—CO, NH—CO—NH,CO—NH—NH—CO, and S—S; Y is selected from the group consisting of NH—CO,CO—NH, CO—NH—NH, NH—NH—CO, NH—CO—NH, CO—NH—NH—CO, and S—S, and the—(CH₂)_(n)—X—(CH₂)Y—Y— moiety is bonded to the 3′, 4′ or 5′ position. Ina preferred embodiment of the hapten-carrier conjugate, m is 11 to 17, nis 1, y is 2, X is NH—CO, Y is CO—NH, the carrier protein is exoproteinA and the —(CH₂)_(n)—X—(CH₂)_(y)—Y— moiety is bonded to the 3′ position.In another preferred embodiment of the hapten-carrier conjugate, m is 11to 17, n is 1, y is 2, X is NH—CO, Y is CO—NH, the carrier protein isexoprotein A and the —(CH₂)_(n)—X—(CH₂)_(y)—Y— moiety is bonded to the4′ position. In a further preferred embodiment of the hapten-carrierconjugate, m is 11 to 17, n is 1, y is 2, X is NH—CO, Y is CO—NH, thecarrier protein is exoprotein A and the —(CH₂)_(n)—X—(CH₂)_(y)—Y— moietyis bonded to the 5′ position. In an additionally preferred embodiment, mis selected from the group consisting of 1 to 20 and 1 to 200.

[0017] The above objects also be achieved by providing a hapten-carrierconjugate of formula (III):

[0018] wherein n is 0 to 12, j is 1 to 1000, k is 1 to 20, and E is anamino acid-containing matrix. In a preferred embodiment, the matrix ispoly-L-glutamic acid.

[0019] The objects can also be achieved by providing an antibody whichis produced in response to the hapten-carrier conjugate of Formula (I).In an additional embodiment, the antibody is a functional fragment. In apreferred embodiment, the antibody is a monoclonal antibody. In anadditional embodiment of the invention, the antibody is polyclonal.

[0020] The objects can also be achieved by providing an antibody whichis produced in response to the hapten-carrier conjugate of Formula(III). In an additional embodiment, the antibody is a functionalfragment. In a preferred embodiment, the antibody is a monoclonalantibody. In an additional embodiment of the invention, the antibody ispolyclonal.

[0021] The objects can be achieved by providing a method of treating orpreventing nicotine addiction in a patient in need of such treatmentcomprising administering a therapeutically effective amount of thehapten-carrier conjugate of Formula (I) or (III). Alternatively, theobjects can be achieved by providing a method treating or preventingnicotine addiction in a patient in need of such treatment comprisingadministering a therapeutically effective amount of antibody raised inresponse to the hapten-carrier conjugates of Formula (I) or (III).

[0022] Additionally, the objects can be achieved by providing a vaccinecomposition which comprises the hapten carrier conjugate of Formula (I)or Formula (III). In addition the vaccine can further comprise anadditional therapeutic compound for treating nicotine addiction.

[0023] The objects also can be achieved by providing a process forproducing an antibody, comprising immunizing a host mammal with ahapten-carrier conjugate of Formula (I) or (III). In a preferredembodiment, the antibody produced is a monoclonal antibody. In anadditional embodiment the antibody is polyclonal.

[0024] Additional objects can be achieved by providing a kit fordetermining the presence of nicotine in a sample, comprising an antibodyof raised in response to the hapten-carrier conjugate of Formula (I) orFormula (III).

[0025] These objects and others apparent to those skilled in the arthave been achieved by the invention described below in the detaileddescription and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 is a chart that shows the effect of active immunization,with a 3′AMNic-Suc-rEPA conjugate vaccine, on nicotine blood serumlevels in rats, following a singe injection of nicotine. Nicotine serumlevels, 3 and 10 minutes after nicotine injection, are shown.

[0027]FIG. 2 shows the effect of passive immunization, with antibodiesagainst 3′-AMNic-Suc-rEPA, on nicotine levels in blood and brain ofrats. Rats were treated with 12.5, 25 and 50 mg of antibody.

[0028]FIG. 3 shows the effects of passive immunization, with antibodiesagainst 3′-AMNic-Suc-rEPA, on nicotine levels in blood serum and brain,in rats. Nicotine levels were measured 30 minutes and 1 day afterantibody administration and 3 minutes for nicotine injection.

[0029]FIG. 4 shows the effect of passive immunization, with antibodiesagainst 3′-AMNic-Suc-rEPA, on nicotine blood serum levels, in ratsreceiving multiple doses of nicotine.

[0030]FIG. 5 shows the effects of passive immunization, with antibodiesagainst 3′-AMNic-Suc-rEPA, on nicotine levels in rat brain, in ratsreceiving multiple doses of nicotine.

[0031]FIG. 6 shows the effects of passive immunization, with antibodiesagainst 3′-AMNic-Suc-rEPA, on nicotine-induced locomoter effects, inrats.

[0032]FIG. 7 shows the effects of passive immunization, with antibodiesagainst 3′-AMNic-Suc-rEPA, on nicotine-induced increase in systolicblood pressure. The Figure shows that the increasing amounts of antibodyincreases the effectiveness of the antibodies in decreasing thenicotine-increase in blood pressure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The present invention provides a nicotine hapten-carrierconjugate for treating addiction to nicotine. The nicotinehapten-carrier conjugate is of formula (I):

[0034] wherein m is 1 to 2,500; n is 0 to 12; y is 1 to 12; X isselected from the group consisting of NH—CO, CO—NH, CO—NH—NH, NH—NH—CO,NH—CO—NH, CO—NH—NH—CO and S—S; Y is selected from the group consistingof NH—CO, CO—NH, CO—NH—NH, NH—NH—CO, NH—CO—NH, CO—NH—NH—CO and S—S; thecarrier protein is any suitable immunogenic protein or polypeptide.Preferably the carrier protein may comprise a T-cell epitope, and the—(CH₂)_(n)—X—(CH₂)_(y)—Y— moiety is bonded to the 3′, 4′ or 5′ positionof the nicotine molecule.

[0035] In formula (I), m is preferably 1 to 200. In another preferredembodiment, m is 1 to 20. In a particularly preferred embodiment, m is11 to 17. In another preferred embodiment, X is selected from the groupconsisting of NH—CO, CO—NH, CO—NH—NH, NH—NH—CO, NH—CO—NH, andCO—NH—NH—CO.

[0036] If m is more than one, the moiety in brackets is attached m timesto different points of attachment in the carrier protein. For example,if m=2, then formula (I) would be:

[0037] Because antibodies cannot be raised in response to nicotineitself, the present inventors have developed a nicotine hapten which isderivatized at the 3′, 4′, or 5′ position of nicotine. This moiety isbound to a carrier protein to yield a hapten carrier conjugate, whichwill raise antibodies against the nicotine moiety, when it is injectedinto a suitable host mammal. In this regard, in order for apharmaceutical composition comprising the hapten carrier conjugate toinduce the production of antibodies when administered to a mammal, thecarrier protein must be immunogenic. Preferably, it will comprise a Tcell-epitope. Thus, when the carrier protein is conjugated to thenicotine hapten, and subsequently is administered to a mammal, themammal produces, or “raises” antibodies in response to the nicotinehapten.

Haptens and Derivatization

[0038] The term “hapten” as used in the present invention refers to alow-molecular weight organic compound that is not capable of elicitingan immune response by itself but will elicit an immune response onceattached to a carrier molecule. In a preferred embodiment, the hapten isattached to the carrier via a linker. A hapten of the present inventionis a nicotine derivative. This nicotine hapten contains a reactivefunctional group, to which the carrier can be attached directly, or viaa linker, or via a matrix, or via a linker and a matrix. Preferably, thenicotine hapten is attached to the carrier protein via an amide ordisulfide bond. Amide and disulfide bonds have the desirable property ofstability. Because the hapten-carrier conjugates of the invention willbe used as vaccines, it is important that the conjugates are stable, toprolong the shelf life of the vaccine.

[0039] In a preferred embodiment of the present invention, the nicotinehapten is represented by formula (11):

[0040] wherein n is 0 to 12 and Z is NH₂, COOH, CHO or SH and—(CH₂)_(n)-Z can be bonded to the 3′, 4′ or 5′ position. The Z moiety iscapable of binding to a carrier, directly or via a linker. Thecarrier-hapten conjugate will induce the production of antibodies uponits introduction into the body of a patient or an animal.

[0041] In a particularly preferred embodiment, the nicotine hapten is ofthe following formula (3′-aminomethyl nicotine):

[0042] 1. Direct Conjugates

[0043] To make a “direct conjugate,” a single nicotine hapten isdirectly attached to a carrier, with or without a linker. For example, asingle nicotine hapten can be attached to each available amine group onthe carrier. General methods for directly conjugating haptens to carrierproteins, using a homobifunctional or a heterobifunctional cross-linkerare described, for example, by G. T. Hermanson in BioconjugateTechniques, Academic Press (1996) and Dick and Beurret in ConjugateVaccines. Contribu. Microbiol. Immunol., Karger, Basal (1989) vol. 10,48-114. With direct conjugation using bifunctional crosslinkers, themolar ratio of hapten to protein is limited by the number of functionalgroups available on the protein for the specific conjugation chemistry.For example, with a carrier protein possessing n number of lysinemoieties, there will be, theoretically, n+1 primary amines (includingthe terminal amino) available for reaction with the linker's carboxylicgroup. Thus, using this direct conjugation procedure the product will belimited to having n+1 amido bonds formed, i.e., a maximum of n+1 haptensattached.

[0044] The skilled artisan will recognize that depending on theconcentration of the reactants used to conjugate the nicotine hapten tothe carrier protein, and the nature of the carrier protein, the ratio ofhapten to carrier will vary. Also, within a given preparation ofnicotine-carrier conjugate, there will be variation in thehapten/carrier ratio of each individual conjugate. For example,exoprotein A has, in theory, 15 amines available for conjugation withhapten. However, the inventors determined that when3′aminomethyl-succinyl-nicotine was conjugated to this protein, a rangeof 11-17 nicotine haptens were attached to each exoprotein A carrier, ina single preparation of conjugate. This range was experimentallydetermined using gas filtration chromatography and measuring theincrease in UV absorbance at 260 nm. 17 nicotines were attached to somecarriers because the nicotine hapten can attach to non-amine moieties onthe carrier. Examples of non-amine moieties to which the hapten canattach include, but are not limited to, —SH and —OH moieties. However,the incidence of these side reactions is low.

[0045] 2. Matrix Conjugates

[0046] To circumvent the limitations on the number of haptens that canbe attached to carrier using direct conjugation, an amino acid “matrix”can be used. The term “matrix” denotes an amino acid, a peptide,dipeptide, or a polypeptide, including oligomeric and polymericpolypeptides. A matrix also may be a linear or branched polypeptide.Examples of amino acids that may be used to form a matrix include, butare not limited to, aspartic acid, lysine, cysteine, and L-glutamicacid. Such matrix materials may be formulated into polymers, such aspoly-L-glutamic acid. When an amino acid such as cysteine is used, thethiol group is protected, thereby permitting the hapten to be linked tothe carboxylic group of the amino acid. One skilled in the art would bewell familiar with types of protecting groups and means of attachingprotecting groups to amino acid functionalities. For a discussion, seeGreen, PROTECTIVE GROUPS IN ORGANIC CHEMISTRY, John Wiley & Sons, NewYork, 1991.

[0047] A suitable matrix possesses an appropriate functional group andis loaded with two or more haptens. Thus, in another preferredembodiment of the invention, the nicotine-substituted matrix isconjugated to the carrier protein to increase the hapten to carriermolar ratio in the hapten-carrier conjugate. The matrix plays a doublerole, first, as a support for a large number of haptens and, second, asa cross linker. The nicotine substituted matrix conjugated to a carrierprotein is represented by formula (III):

[0048] wherein n is 0 to 12, j is 1 to 1000, k is 1 to 20 E is an aminoacid-containing matrix to which a hapten can be bonded, and the carrierprotein is any suitable protein or polypeptide comprising a T-cellepitope. The amino acid-containing matrix E may be an amino acid, apeptide, dipeptide, or a polypeptide, including oligomeric as well aspolymeric polypeptides. The matrix comprises one or more amino acidsthat include, but are not limited to, aspartic acid, lysine, cysteine,and poly-L-glutamic acid. In a preferred embodiment, j is 1 to 200, andin another preferred embodiment, j is 1 to 4.

[0049] Matrix-carrier conjugates are capable of forming multimeric“lattices.” Such a lattice is represented in the figure below. The term“lattice” is used to denote a covalently-linked complex, comprisingmultiple matrices, haptens, linkers and carrier proteins, all of whichare covalently linked together. Because the nicotine-substituted matrixcomprises multiple nicotine moieties available for conjugation withcarrier, a lattice comprising multiple carriers, and multiplenicotine-substituted matrices, can be formed. A simplifiedrepresentation of a portion of such a lattice is represented as follows:

[0050] The skilled artisan will recognize that a lattice according tothe invention comprises a hapten carrier conjugate of Formula (III).

[0051] This conjugation method employing a matrix offers flexibility andcontrol over hapten to protein molar ratios regardless of the number offunctional groups available for conjugation on the protein. This isparticularly useful when a specific carrier protein has been used andwhen an optimal ratio needs to be obtained in order to achieve higherimmunogenicity of the conjugate. While it is not necessary to use anwhen using a matrix, such a linker can be used. To use a linker withthis embodiment, the nicotine substituted matrix is reacted with anactive linker compound. For example, ADH, adipic acid dihydrazide, canbe used as a linker with the matrix conjugates.

Carrier Proteins

[0052] Once the nicotine hapten has been prepared, it is then conjugatedto a carrier protein which will be used to raise antibodies to thenicotine carrier conjugate. The carrier protein used in the presentinventive nicotine carrier conjugate is represented by

[0053] in formulae (I) and (III) and encompasses any suitableimmunogenic protein or polypeptide. An “immunogenic” molecule is onethat is capable of eliciting an immune response. Preferably, the carrierprotein will comprise a T-cell epitope. Also encompassed by therepresentation of a “carrier protein” are MAPs or multi-antigenicpeptides, which are branched peptides. By using a MAP, hapten densityand valency are maximized because of multiple branched amino acidresidues. Examples of amino acids that can be used to form a MAPinclude, but are not limited to, lysine.

[0054] A carrier protein of the instant invention comprises a moleculecontaining at least one T cell epitope which is capable of stimulatingthe T cells of the subject, which subsequently induces B cells toproduce antibodies against the entire hapten-carrier conjugate molecule.The term “epitope” as used in describing this invention, includes anydeterminant on an antigen that is responsible for its specificinteraction with an antibody molecule. Epitopic determinants usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains and have specific three dimensionalstructural characteristics as well as specific charge characteristics.It is believed that to have immunogenic properties, a protein orpolypeptide must be capable of stimulating T-cells. However, it ispossible that a carrier protein that lacks a T-cell epitope may also beimmunogenic.

[0055] By selecting a carrier protein which is known to elicit a strongimmunogenic response, a diverse population of patients can be treated bythe inventive hapten-carrier conjugates. The carrier protein must besufficiently foreign to elicit a strong immune response to the vaccine.Typically, the carrier protein used preferably would be a large moleculethat is capable of imparting immunogenicity to a covalently-linkedhapten. A particularly preferred carrier protein is one that isinherently highly immunogenic. Thus a carrier protein that has a highdegree of immunogenicity and is able to maximize antibody production tothe hapten is highly desirable.

[0056] Both bovine serum albumin (BSA) and keyhole limpet hemocyanin(KLH) have commonly been used as carriers in the development ofconjugate vaccines when experimenting with animals. However, theseproteins may not be suitable for human use. Proteins which have beenused in the preparation of therapeutic conjugate vaccines include, butare not limited to, a number of toxins of pathogenic bacteria and theirtoxoids. Examples include diphtheria and tetanus toxins and theirmedically acceptable corresponding toxoids. Other candidates areproteins antigenically similar to bacterial toxins referred to ascross-reacting materials (CRMs).

[0057] In the preparation of nicotine conjugate pharmaceuticalcompositions, recombinant Pseudomonas aeruginosa exoprotein A (rEPA) maybe used as a carrier protein because its structure and biologicalactivities have been well characterized. Moreover, this recombinantprotein has been successfully and safely used in humans in theStaphylococcus aureus capsular polysaccharide conjugate vaccines by theNational Institutes of Health and by the present inventors. Fattom etal., Infect Immun. 61 1023-1032 (1993). This protein has been identifiedas a suitable protein carrier because the intrinsic enzymatic activityof the native exotoxin has been eliminated due to an amino acid deletionat position 553. As a result, rEPA has the same immunological profile asthe native exotoxin A (ETA), but does not possess the hepatotoxicproperties of the native ETA. As used in this application, “exoproteinA” refers to a modified, non-hepatotoxic, ETA. On example of such anexoprotein A has an amino acid deletion at position 553.

Conjugation of Hapten to Carrier Protein

[0058] There are a large number of functional groups which can be usedin order to facilitate the linking or conjugation of a carrier to asmall molecule, such as a hapten. These include functional moieties suchas carboxylic acids, anhydrides, mixed anhydrides, acyl halides, acylazides, alkyl halides, N-maleimides, imino esters, isocyanates, amines,thiols, and isothiocyanates and others known to the skilled artisan.These moieties are capable of forming a covalent bond with a reactivegroup of a protein molecule. Depending upon the functional moiety used,the reactive group may be the E amino group of a lysine residue or athiol group, on a carrier protein or a modified carrier protein moleculewhich, when reacted, results in amide, amine, thioether, amidine urea orthiourea bond formation. One skilled in the art would recognize thatother suitable activating groups and conjugation techniques can be used.See, for example, Wong, Chemistry of Protein Conjugation andCross-Linking, CRC Press, Inc. (1991). See also Hermanson, BIOCONJUGATETECHNIQUES, Academic Press: 1996 and Dick and Beurret in ConjugateVaccines. Contribu. Microbiol. Immunol., Karger, Basal (1989) vol. 10,48-114.

[0059] Linear linker moieties are preferred, over cyclic or branchedlinkers, for conjugation of haptens to carrier proteins. A preferredlinker is a succinyl moiety. However, a linker may be a cyclic structureas well as a linear moiety. Another example of a linker is ADH.

[0060] Thus, the nicotine hapten-carrier conjugates of the presentinvention are prepared by reacting one or more haptens with a carrierprotein to yield a hapten carrier conjugate which is capable ofstimulating T cells, leading to T cell proliferation and release ofmediators which activate specific B cells to stimulate antibodyproduction in response to the immunogenic hapten-carrier conjugate.Certain antibodies raised in response to the hapten carrier conjugatewill be specific to the hapten portion of the hapten-carrier conjugate.The present invention contemplates the use of various suitablecombinations of haptens with carrier proteins for use in the treatmentof nicotine addiction.

Monoclonal and Polyclonal Antibodies

[0061] Techniques for making monoclonal antibodies are well-known in theart. Monoclonal antibodies can be obtained by injecting mice with acomposition comprising the nicotine hapten-carrier conjugate,subsequently verifying the presence of antibody production by removing aserum sample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to thehapten-carrier conjugate, culturing the clones that produce antibodiesto the antigen, and isolating the antibodies from the hybridomacultures.

[0062] Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Inununoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

[0063] Techniques for preparing polyclonal antibodies also arewell-known in the art. Polyclonal antibodies are prepared according tostandard techniques known in the art. To prepare a polyclonal antibody,an animal is injected with the immunogenic material and antibody richserum is collected which contains therein a mixture of antibodies thatare directed against numerous epitopes of the immunogen that wasinjected. Suitable host mammals for the production of antibodiesinclude, but are not limited to, humans, rats, mice, rabbits, and goats.

[0064] In accordance with the present invention, functional antibodyfragments also can be utilized. The fragments are produced by methodsthat include digestion with enzymes such as pepsin or papain and/orcleavage of disulfide bonds by chemical reduction. Alternatively,antibody fragments encompassed by the present invention can besynthesized using an automated peptide synthesizer such as thosesupplied commercially by Applied Biosystems, Multiple Peptide Systemsand others, or they may be produced manually, using techniques wellknown in the art. See Geysen et al., J. Immunol. Methods 102: 259(1978). Direct determination of the amino acid sequences of the variableregions of the heavy and light chains of the monoclonal antibodiesaccording to the invention can be carried out using conventionaltechniques.

[0065] A fragment according to the present invention can be an Fvfragment. An Fv fragment of an antibody is made up of the variableregion of the heavy chain (Vh) of an antibody and the variable region ofthe light chain of an antibody (V1). Proteolytic cleavage of an antibodycan produce double chain Fv fragments in which the Vh and V1 regionsremain non-covalently associated and retain antigen binding capacity. Fvfragments also include recombinant single chain antibody molecules inwhich the light and heavy chain variable regions are connected by apeptide linker. See Skerra, et al. Science, 240, 1038-41 (1988).Antibody fragments according to the invention also include Fab, Fab′,F(ab)₂, and F(ab′)₂, which lack the Fc fragment of an intact antibody.

Therapeutic Methods

[0066] Because nicotine exerts many of its significant effects after itcrosses the blood brain barrier, the present invention encompassestherapeutic methods that prevent nicotine from crossing the blood brainbarrier. In particular, administration of a nicotine hapten-carrierconjugate to a patient will generate antibodies against nicotine, in thebloodstream of the patient. Alternatively, anti-nicotine antibodiesgenerated outside the body of the patient to be treated, in a suitablehost mammal, can be administered to a patient. If the patient smokes,the nicotine in his blood will be bound by the circulating anti-nicotineantibodies, preventing the nicotine from reaching the brain. Therefore,the antibodies will prevent the physiological and psychological effectsof nicotine that originate in the brain. Because the smoker willexperience a lessening or cessation of these effects, he/she will losethe desire to smoke. The same therapeutic effects are expected if apatient uses smokeless tobacco, after being immunized with a nicotinehapten-carrier conjugate of the invention. Additionally, the conjugatesand antibodies of the invention may exert their effects by affecting theability of nicotine to stimulate the peripheral nervous system.

[0067] As discussed above, the novel nicotine-carrier conjugates of theinvention preserve the native chirality and structure of the nicotinemolecule. In particular, the nicotine moiety of these conjugates has the(S)-(−) configuration. Therefore, the antibodies produced in response tosuch a conjugate will be specific to the native form of nicotine, andwill be the most effective in specifically binding to nicotine that isinhaled from smoking or absorbed from smokeless tobacco, and ininhibiting the effects of this ingested nicotine. Additionally, theinventive conjugates are chemically stable, and stability is critical toproducing a vaccine having a long shelf life.

[0068] The present vaccine composition can be used in combination withcompounds or other therapies that are useful in the treatment ofaddiction. This includes administration of compounds which include, butare not limited to, anti-depressant drugs such as Zyban and Prozac.

[0069] 1. Administration of a Nicotine Hapten-Carrier Conjugate

[0070] The conjugates of the invention are suitable for treating andpreventing nicotine addiction. For treating nicotine addiction, anicotine-carrier conjugate of the invention is administered to a patientsuffering from nicotine addiction. For preventing nicotine addiction,patients at risk for developing nicotine addiction, such as teenagers,are treated with a conjugate according to the invention. Directadministration of the conjugate to a patient is called “activeimmunization.”

[0071] A vaccine composition of the present invention comprises at leastone nicotine hapten-carrier conjugate in an amount sufficient to elicitan immune response thereto. The nicotine hapten carrier conjugate iscapable of remaining in vivo at a concentration sufficient to be activeagainst subsequent intake of nicotine.

[0072] Initial vaccination with the nicotine hapten carrier conjugate ofthe present invention creates high titers of antibodies that arespecific to nicotine. The therapeutically effective amount of aconjugate which is administered to a patient in need of treatment fornicotine addiction is readily determined by the skilled artisan.Suitable dosage ranges are 1-1000 μg/dose. It generally takes a patientone to several weeks to generate antibodies against a foreign antigen.The production of antibodies in a patient's blood can be monitored byusing techniques that are well-known to the skilled artisan, such asELISA, radioimmunoassay, and Western blotting methods. Therapeuticeffectiveness also can be monitored by assessing various physicaleffects of nicotine, such as blood pressure.

[0073] As described in detail below, the inventive nicotinehapten-carrier conjugates can be processed to afford a composition whichcan be readily administered to a patient. The preferred modes ofadministration include but are not limited to intranasal, intratracheal,oral, dermal, transmucosal subcutaneous injection and intravenousinjection. The skilled artisan will recognize that the initial injectionmay be followed by subsequent administration of one or more “boosters”of conjugate. Such a booster will increase the production of antibodiesagainst the nicotine hapten-carrier conjugate of the invention.

[0074] The vaccine compositions of the present invention may contain atleast one adjuvant. The adjuvant used in the present invention will beselected so that the effect of the carrier protein is not inhibited.Adjuvants used in the present invention are those which arephysiologically acceptable to humans, these include, but are not limitedto, alum, QS-21, saponin and MPLA (monophosphoryl lipid A).

[0075] The vaccine compositions of the present invention may optionallycontain one or more pharmaceutically acceptable excipients. Theexcipients useful in the present include sterile water, salt solutionssuch as saline, sodium phosphate, sodium chloride, alcohol, gum arabic,vegetable oils, benzyl alcohols, polyethylene glycol, gelatin, mannitol,carbohydrates, magnesium stearate, viscous paraffin, fatty acid esters,hydroxy methyl cellulose and buffers. Of course, any additionalexcipients known to the skilled artisan are useful in the presentinvention.

[0076] The hapten-carrier conjugates of the present invention, in orderto be administered to a patient in need of treatment or prevention ofnicotine addiction, are incorporated into a pharmaceutical composition.When the composition containing the hapten-carrier conjugate is to beused for injection, it is preferable to solubilize the hapten-carrierconjugate in an aqueous, saline solution at a pharmaceuticallyacceptable pH. However, it is possible to use an injectable suspensionof the hapten-carrier conjugate. In addition to the usualpharmaceutically acceptable excipients, the composition may containoptional components to ensure purity, enhance bioavailability and/orincrease penetration.

[0077] Additionally, the vaccine composition may optionally contain atleast one auxiliary agent, such as dispersion media, coatings,microspheres, liposomes, microcapsules, lipids, surfactants, lubricants,preservatives and stabilizers. Of course, the any additional auxiliaryagents known to the skilled artisan are useful in the present invention.Also useful herein are any agents which act to synergize the effect ofthe present vaccine composition.

[0078] The pharmaceutical composition of the present invention issterile and is sufficiently stable to withstand storage, distribution,and use. Additionally, the composition may contain additional componentsin order to protect the composition from infestation with, and growthof, microorganisms. It is preferred that the composition is manufacturedin the form of a lyophilized powder which is to be reconstituted by apharmaceutically acceptable diluent just prior to administration.Methods of preparing sterile injectable solutions are well known to theskilled artisan and include, but are not limited to, vacuum drying,freeze-drying, and spin drying. These techniques yield a powder of theactive ingredient along with any additional excipient incorporated intothe pre-mix.

[0079] 2. Administration of Antibodies Produced in Response to aNicotine-Carrier Conjugate

[0080] Passive immunization comprises administration of or exposure to apolyclonal antibody or monoclonal antibody which has been raised inresponse to a nicotine hapten carrier conjugate of the invention. Suchantibodies can be generated in animals or humans. Antibodies raised inresponse to a nicotine conjugate of the invention can be administered toprevent addiction to nicotine. For example, such antibodies can beadministered to people considered to be at risk for developing addictionto nicotine, such as teenagers. Antibodies also are suitable fortreating a patient addicted to nicotine. As discussed above, theantibodies will bind nicotine in the blood, and prevent nicotine fromcrossing the blood brain barrier. Antibodies raised by administration ofthe inventive hapten-carrier conjugate have a molecular weight range offrom about 150 kDa to about 1,000 kDa.

[0081] The therapeutically effective amount of a therapeutic antibody ofthe invention which is administered to a patient in need of treatmentfor nicotine addiction is readily determined by the skilled artisan.Suitable dosage ranges are 1-1000 μg/dose.

[0082] A therapeutic composition of the present invention comprises atleast antibody produced in response to a nicotine-carrier conjugate ofthe invention. These compositions of the present invention mayoptionally contain one or more pharmaceutically acceptable excipients.The excipients useful in the present include sterile water, saltsolutions such as saline, sodium phosphate, sodium chloride, alcohol,gum arabic, vegetable oils, benzyl alcohols, polyethylene glycol,gelatin, mannitol, carbohydrates, magnesium stearate, viscous paraffin,fatty acid esters, hydroxy methyl cellulose and buffers. Of course, anyadditional excipients known to the skilled artisan are useful in thepresent invention.

[0083] The antibodies of the present invention, in order to beadministered to a patient in need of treatment or prevention of nicotineaddiction, are incorporated into a pharmaceutical composition. When thecomposition containing an antibody is to be used for injection, it ispreferable to have the antibody in an aqueous, saline solution at apharmaceutically acceptable pH. However, it is possible to use aninjectable suspension of the antibody. In addition to the usualpharmaceutically acceptable excipients, the composition may containoptional components to ensure purity, enhance bioavailability and/orincrease penetration.

[0084] A pharmaceutical composition comprising an antibody of thepresent invention is sterile and is sufficiently stable to withstandstorage, distribution, and use. Additionally, the composition maycontain additional components in order to protect the composition frominfestation with, and growth of, microorganisms. Methods of preparingsterile injectable solutions are well known to the skilled artisan andinclude, but are not limited to, vacuum drying, freeze-drying, and spindrying. These techniques yield a powder of the active ingredient alongwith any additional excipient incorporated into the pre-mix.

Kits Comprising Antibodies of the Invention

[0085] The antibodies of the present invention also are useful inpreparing a kit that can be used to detect and quantify nicotine levelsin a sample. A kit according to the invention comprises anicotine-specific antibody according to the invention, in a suitablecontainer. For a radioimmunoassay, the kit may also comprise labelednicotine. Nicotine in a sample is detected by binding labeled nicotineto the antibody, and then competing the labeled nicotine from theantibody with the sample to be tested. An ELISA kit also would comprisean antibody according to the invention. The ELISA may involve inhibitionof antibody binding with known amounts of nicotine compared toinhibition with a sample suspected of containing nicotine. This wouldallow determination of unknown nicotine in a sample, by comparison ofsample with the standard inhibition curve of known nicotineconcentration. In another type of ELISA, a sample suspected ofcontaining nicotine would be incubated with a microtiter plate that hasbeen coated with a substance that will bind nicotine. The antibodies ofthe invention would be added, and enzyme-linked anti-antibody antibodieswould be added to the plates. Addition of substrate would quantify theamount of nicotine bound to the plate.

[0086] The following examples are provided merely to further illustratethe preparation and use of the present invention. The scope of theinvention is not limited to the following examples.

EXAMPLE 1

[0087] Synthesis of a Derivitized Nicotine Hapten (Substituted at the 3′Position)

[0088] The starting material for the synthesis of the hapten istrans-4′-carboxy-(−)-cotinine, available from commercial sources. Amodification of the procedure described by Cushman and Castagnoli, Jr.(1972) J. Org. Chem. 37(8):1268-1271 provides the alcohol,trans-3′-hydroxymethyl-(−)-nicotine, after methyl esterification of theacid followed by reduction of the ester. The alcohol is then sulfonatedand the sulfonate is displaced with an azido group, which is finallyreduced to an amine.

[0089] 4 g of trans-4′-carboxy-(−)-cotinine are dissolved in 50 mL of asolution of 2 N sulfuric acid in dry methanol and stirred overnight atroom temperature. The resulting suspension is filtered through a WhatmanNo. 1 filter paper and added slowly to 100 mL of a saturated solution ofsodium bicarbonate. The ester is extracted with dichloromethane toafford 4.2 g of a pink oil after solvent evaporation.

[0090] A solution of 3.9 g of the ester in dry tetrahydrofuran (100 mL)is added dropwise to a suspension of 4 equivalents of lithium aluminumhydride in dry tetrahydrofuran (70 mL) under dry argon. The suspensionis stirred for two hours at room temperature. Excess hydride isdestroyed by careful and controlled addition of water while cooling inan ice bath. The resulting white precipitate is filtered off and thefiltrate dried over sodium sulfate and concentrated under reducedpressure to afford 2.7 g of the alcohol as a yellow oil.

[0091] The alcohol (1.9 g) is dissolved in 20 mL of dichloromethane.Triethylamine (0.75 mL) and p-toluenesulfonyl chloride(l g) are thensuccessively added to the solution. The orange solution is stirred for24 hours at room temperature. Precipitated triethylamine hydrochlorideis filtered off on a Celite bed and the filtrate is concentrated underreduced pressure to give a brown oil. The sulfonate is purified on asilica flash chromatography column eluted with 5% methanol indichloromethane to give 2.1 g of a yellow oil.

[0092] The sulfonate (1.8 g) is displaced using sodium azide (0.8 g) in50 mL dimethylformamide for one hour at 80° C. After evaporation ofdimethylformamide under high vacuum, the residue is dissolved indichloromethane, washed with water and brine and dried over sodiumsulfate. After solvent evaporation, the azide (1.1 g) is obtained as abrownish oil.

[0093] The addition of the azide in dry tetrahydrofuran (20 mL) to asuspension of lithium aluminum hydride in dry tetrahydrofuran (50 mL)readily produced the desired amine as monitored by thin layerchromatography. Proton and carbon nuclear magnetic resonance spectra ofthe purified amine corresponded to the expected structure.

EXAMPLE 2 Synthesis of a Derivitized Nicotine Hapten (Substituted at the4′ Position)

[0094] Introduction of a functionalized arm on position 4′ of nicotinecan be achieved by enolate alkylation of cotinine followed by reductionof the alkylated product. Various alkylating agents can be used like anappropriately protected 3-bromo-propylamine. As examples,3-bromo-N-carbobenzyloxy-propylamine or N-(3-bromopropyl)-phtalamide canbe used. The amine protecting group will have to be removed afteralkylation and reduction and prior to conjugation to a carrier protein.Enolate alkylation of cyclic lactams (containing the pyrrolidinone ring)is well documented in the literature (see G. Helmchen et al. (1995)Steroselective Synthesis in Houben-Weyl-Methods of Organic Chemistry,Vol. E21a, 762-881, Thieme, Stuttgart, Germany, for a general review,and A. J. Meyers et al. (1997) J. Am. Chem. Soc., 119, 4564-4566, forsteric considerations of the reaction). There are also some examples ofenolate alkylation of cotinine itself (N. -H. Lin et al. (1994) J. Med.Chem., 37, 3542-3553). An interesting preparation of 4′-acetyl-nicotine,as a 1:1 mixture of two epimers, was achieved using a tandem cationicaza-Cope rearrangement-Mannich cyclization reaction starting from aketone (or an aldehyde) and a 2-alkoxy-3-alkenamine (L. E. Overman(1983) J. Am. Chem. Soc., 105, 6622-6629). This reaction can be extendedto produce 4′-aldehydo-nicotine, suitable for conjugation.

[0095] 3-bromo-propylamine hydrobromide (4.2 g) was suspended in 50 mLdichoromethane and triethylamine (about 7 mL) was added until a clearsolution was obtained. The solution was cooled to 0° C. and benzylchloroformate (2.5 mL) was added dropwise. The reaction was allowed toproceed at room temperature for 16 hours under stirring. Theprecipitating salts were filtered off and the clear organic layer waswashed with cold water, cold 1 N HC1 and cold water, dried on sodiumsulfate and evaporated under reduced pressure to a yellow oil (2.93 g ofcrude material).

[0096] Cotinine (62mg) and 3-bromo-N-carbobenzyloxy-propylamine (100 mg)were separately co-evaporated with dry toluene. Cotinine was dissolvedin 5 mL of freshly distilled anhydrous tetrahydrofuran, 60 μL ofN,N,N′,N′-tetramethylenediamine (TMEDA) were added and the solutioncooled to −78° C. by immersion in an ethanol-dry ice bath. The cotininesolution was added dropwise to a solution of lithium diisopropylamide(LDA, 200 μL of a 2 M solution in heptane-tetrahydrofuran) intetrahydrofuran, previously cooled to −78° C. The orange mixture isstirred for 15 minutes at −78° C. and then left to warm up in an icebath (2 to 6° C.). The reaction was then cooled again to −78° C. and3-bromo-N-carbobenzyloxy-propylamine dissolve in anhydroustetrahydrofuran added dropwise for 15 minutes. The reaction mixture wasleft to warm-up to −10° C. and then quenched with methanol. The reactionproduct was purified by flash chromatography on a silica gel column.Reduction of the amide of this cotinine derivative was achieved withborane followed by cesium fluoride in hot ethanol. The final amine wasobtained after removal of the carbobenzyloxy group in acidic conditions.

EXAMPLE 3 Synthesis of a Derivitized Nicotine Hapten (Substituted at the5′ Position)

[0097] Introduction of a functionalized arm on position 5′ of nicotinecan be achieved by reacting appropriately protected alkyl lithiumcompounds with cotinine, followed by reduction with sodiumcyanoborohydride, in procedures similar to those described by Shibagakiet al. (1986) Heterocycles, 24, 423-428 and N. -H. Lin et al. (1994)supra.

EXAMPLE 4 Conjugation of a Derivitized Nicotine Hapten to a CarrierProtein

[0098] Recombinant exoprotein A (rEPA) is linked to the derivitizednicotine hapten through a succinic acid arm. The 15 lysines of rEPA werereadily succinylated with succinic anhydride. Then, in a typicalconjugation reaction, a 5 to 10 mg/mL solution of the succinylatedrecombinant exoprotein A (Suc-rEPA) in a 2-(N-morpholino) ethanesulfonicacid (MES) buffer 0.05 M containing 0.15 M NaCl at pH 6.0 was prepared.An equal weight of 3′-aminomethyl-(−)-nicotine (3′AMNic) haptendissolved in a minimal amount of distilled water was added to theprotein solution. The pH of the hapten solution was adjusted to 6.0 with0.1 N HCl before addition. Finally, an equal weight of1-ethyl-3—(3-diethylamino)propyl carbodiimide hydrochloride (EDC) wasadded to the hapten protein mixture and the reaction proceeded for 30min at room temperature while stirring. The thus obtained nicotineconjugate was purified on a Sephadex G-25 column eluted with phosphatebuffer saline (PBS) at pH 7.4. Conjugate recoveries were in the 80 to90% range.

EXAMPLE 5 Conjugation of Nicotine-Loaded Matrix

[0099] This example describes synthesis of a hapten-carrier conjugatecomprising 3′-aminomethyl-(−)-nicotine as a derivitized hapten,recombinant exoprotein A (rEPA) as a carrier protein, adipic aciddihydrazide (ADH) as a linker and poly-L-glutamic acid as a “matrix,” orpolymer support, for the haptens A poly-L-glutamic acid having anaverage molecular weight of 39,900 with a polydispersity of 1.15 and adegree of polymerization of 264 was used in this example. The reactingamounts of hapten and polymer were calculated so that the target degreeof substitution is about 80%. That is, when 80% substitution is reached,about 208 hapten units were conjugated, out of a total 264 repeat unitsin an average molecule of the glutamic acid polymer.

[0100] This nicotine-loaded poly-L-glutamic acid has the followingformula:

[0101] As indicated in the figure, the polyglutamic acid polymercomprises about 52 glutamine residues. This number will vary, dependingon the batch and source of the polyamino acid residue chosen for thematrix. Also, the figure indicates that 4 nicotine haptens for eachrepeating unit. This number will vary depending on the ratio ofreactants used when the matrix and the nicotine hapten are conjugated.

[0102] Following conjugation with the derivitized nicotine, theunreacted carboxylic groups (about 20%) were then derivatized with ADH.When this matrix was conjugated to a carrier, as described in Example 6,the molar ratio of the nicotine-loaded matrix to protein was 1:1. Thus,in this conjugate, the theoretical nicotine hapten to protein molarratio would be 200:1, at the completion of the conjugation reaction.

[0103] The actual ratio of nicotine substitution on the polyglutamicacid was estimated using NMR analysis of the product. The intensity ofthe glutamic acid a-hydrogen peak relative to the four hydrogens of thepyridine ring of the nicotine provide the proportion of nicotineincorporated. The estimated average ratio was 143:1 (nicotine/carrierprotein).

EXAMPLE 6 Preparation of a Nicotine Conjugate Vaccine UsingNicotine-Loaded Matrix

[0104] A. Loading the Nicotine Hapten on the Matrix

[0105] 10 mg of poly-L-glutamic acid salt (Sigma, Cat #P-4761) weredissolved in 2 mL of 0.05 M 2-(N-morpholino)ethansulfonic acid (MES)buffer containing 0.15 M NaCl at pH 6.0. 10 mg of3′-aminomethyl-(−)-nicotine were dissolved in a minimal amount ofdistilled water and the pH of the solution was adjusted to pH 6.0 with0.1N HCl. The nicotine hapten solution was added dropwise to thepolypeptide solution while stirring and was subsequently adjust to a pHof 6.0. 20mg of solid 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC) were then added in three portions to thehapten-polypeptide mixture over a period of 20 minutes. The reaction wasallowed to proceed for one hour at room temperature. The reactionproduct (nicotine-substituted matrix) was dialyzed against three changesof water and lyophilized. 12 mg of nicotine-substituted polyglutamicacid were obtained as a white fluffy material.

[0106] B. Attachment of the Linker to the Nicotine-Substituted Matrix

[0107] 10 mg of nicotine-substituted polyglutamic acid were dissolved in2 mL of MES buffer at pH 6.0. 8 mg of adipic acid dihydrazide (ADH)followed by 10 mg of EDC were added to the solution while stirring. Thereaction was allowed to proceed for 1 hour at room temperature. Theobtained solution was finally dialyzed against three changes of MESbuffer at pH 6.0.

[0108] C. Conjugation to the Carrier Protein

[0109] 10 mg of recombinant exoprotein A (rEPA) were dissolved in 2 mLof 0.05 M MES buffer at pH 5.6 containing 0.15 NaCl. A volume ofADH-bound nicotine-substituted polyglutamic acid solution estimated tocontain 7.5 mg of this derivatized material was added to the proteinsolution. Solid EDC was added to this mixture in three portions over aperiod of 20 minutes while stirring at room temperature. The reactionwas allowed to proceed at room temperature overnight. The resultingconjugate was finally purified on a Sephadex G-25 column, and elutedwith phosphate buffered saline (PBS) at pH 7.4. This produces a purifiedpreparation of conjugate, wherein the conjugate contains only the(S)-(−) form of nicotine.

EXAMPLE 7 Characterization of the Nicotine Carrier Conjugate of Example4

[0110] The purified conjugate vaccine of Example 4 was analyzed on aSuperose 12 size exclusion chromatography column and eluted with PBS atpH 7.4. The hapten to protein molar ratio of 11 to 17 was calculated bydetermining the increase of the UV absorption at 260 nm after theincorporation of nicotine, relative to the absorption at 280 nm. Thisrange was determined by calculating the hapten/carrier protein ratio ofsix separate prepared lots of hapten-carrier conjugate (lot 1: 17.2, lot2: 16.2, lot 3: 13.2; lot 4: 12.0; lot 5: 11.0; lot 6: 17.2). Furtheranalysis to determine this ratio using MALDI-TOF mass spectrometry gaveessentially the same numbers as obtained by UV absorbance difference.The protein concentration of the conjugate vaccine was determined usinga BCA assay. A stability study of the nicotine-carrier conjugate ofexample 4 was carried out. The study used the vaccine vialed in 1 mLglass vials at a concentration of 0.5 mg/mL and the stability of thevialed vaccine was tested at three different temperatures: −70° C., 2 to8° C. and room temperature.

EXAMPLE 8 -Stability of the Nicotine Carrier Conjugate of Example 4

[0111] The conjugation procedure based on the formation of amide bondsbetween hapten-linker-carrier rather than ester bonds appearedbeneficial as observed in the stability of the conjugate for six monthsat −70° C., 2 to 8° C. and even at room temperature. The stability studyconsisted of monitoring and assaying the conjugate vaccine using thefollowing:

[0112] 1.) Visual observation to look for any particulates formed(turbidity, precipitation).

[0113] 2.) Checking for any significant pH change.

[0114] 3.) Size exclusion chromatography profile in combination with UVabsorption at 260 and 280 nm to determine if the ratio of nicotineincorporation changed.

[0115] 4.) Reversed phase chromatography to check for any carrierprotein degradation.

[0116] 5.) SDS PAGE with silver staining looking for any proteolyticcleavage of the conjugated protein.

[0117] The conjugation procedure based on the formation of an amide bondbetween the hapten and the linker as well as between the linker and thecarrier appeared beneficial as observed in the stability of theconjugate for six months at −70° C. and 2 to 8° C.

EXAMPLE 9 Evidence of Immunogenicity of the Carrier Conjugate ofExamples 4 and 6

[0118] Two nicotine hapten-carrier conjugate vaccines were used toimmunize mice, rats, and rabbits.

[0119] A. Animal Tests-polyclonal Antibodies

[0120] Animals were immunized using standard protocols. In mice, threesubcutaneous injections of vaccine were administered, two weeks apart,with test bleeds performed one week following the first and secondinjection, and exsanguination occurring one week following the thirdinjection. Serum samples were evaluated in an ELISA assay, described inExample 10. The ELISA assay utilized 3′ AMNic-pGlu bound to microtiterplates.

[0121] Rats were immunized intraperitoneally with the vaccines threetimes. Injections were given two weeks apart with test bleeds performedone week following the first and second injection. The rats were thenexsanguinated one week after the third injection. Freund's completeadjuvant was used for the first injection, and incomplete Freund'sadjuvant for the subsequent injections. Serum samples were evaluated inan ELISA assay.

[0122] Rabbits were immunized intramuscularly three times, three weeksapart with 100 μg of vaccine. The initial injection contained Freund'sadjuvant, with subsequent injections containing incomplete Freund'sadjuvant. Rabbits were test bled one week following the second and thirdinjections to ensure adequate titers for production bleeding. Ifadequate titer was acquired as measured by ELISA, rabbits were thenplaced on a weekly production bleed schedule (20 to 40 mL serum perrabbit). Antibody titers were monitored over time and animals wereboosted if necessary to restore antibody levels.

[0123] The results of these immunogenicity studies are shown in Tables1-5. Tables 1 and 2 show the results of an immunogenicity study in mice.In Table 1, the conjugate used was3′aminomethyl-(−)-nicotine-succinyl-rEPA (Example 4). In Table 2, theconjugate used was 3-aminomethyl-(−)-nicotine-polyglutamic acid-ADH-rEPA(Example 6). These Tables show generation of high titers of antibodiesthat specifically bind nicotine. Furthermore, these conjugates showedthe ability to induce the booster response.

[0124] Tables 3 and 4 show the results of an immunogenicity study inrats. In Table 3, the conjugate used was3′aminomethyl-(−)-nicotine-succinyl-rEPA (Example 4). In Table 4, theconjugate used was 3-aminomethyl-(−)-nicotine-polyglutamic acid-ADH-rEPA(Example 6). These Tables show generation of high titers of antibodiesthat specifically bind nicotine. Furthermore, these conjugates showedthe ability to induce the booster response.

[0125] Table 5 shows the results of an immunogenicity study in rabbits.Using either 3′aminomethyl-succinyl-rEPA (Example 4) or3-aminomethyl-polyglutamic acid-ADH-rEPA (Example 6), high titers ofantibodies were generated against the two conjugate. Those titersremained elevated for more than 6 months. TABLE 1 Treatment of mice with3′ AMNic-Suc-rEPA Number of Titer Animals Dose 1 injection 2 injections3 injections 10  1 μg 0   36  4,280 10  5 μg 1  884 10,727 10 15 μg 32,476 14,160

[0126] TABLE 2 Treatment of mice with 3′ AMNic-pGlu-ADH-rEPA Number ofTiter Animals Dose 1 injection 2 injections 3 injections 10  2 μg 2  739 6,586 10 10 μg 2 2,490 9,573 10 30 μg 11  2,822 8,195

[0127] TABLE 3 Treatment of rats with 3′AMNic-Suc-rEPA Number of TiterAnimals Dose 1 injection 2 injections 3 injections 3 15 μg  18 7,9429,947 3 25 μg  4 1,446 5,991 3 50 μg 353 7,211 8,996

[0128] TABLE 4 Treatment of rats with 3 ′AMNic-pGlu-ADH-rEPA Number ofTiter Animals Dose 1 injection 2 injections 3 injections 5 100 μg 01,067 3,752

[0129] TABLE 5 Treatment of rabbits with 3′AMNic-Suc-rEPA and3′AMNic-pGlu-ADH-rEPA Number of Immunogen Animals Dose Titer3′-AMNic-Suc-rEPA 10 100 μg 132,000 3′AMNic-pGLu-ADH-rEPA 10 100 μg147,000

EXAMPLE 10 ELISA Assay and Antibody Specificity

[0130] The nicotine molecule itself is not suitable for coating ELISAplates and needs to be linked to a larger molecule having betteradhesive properties. Poly-L-lysine or poly-L-glutamic acid are commonlyused for this purpose. The derivitized nicotine hapten3′-aminomethyl-(−)-nicotine (3′AMNic) was conjugated to poly-L-glutamicacid and the 3′-aminomethyl-(−)-nicotine-poly-L-glutamic acid conjugate(3′AMNic-pGlu ) obtained was used to coat the ELISA plates.

[0131] Antibodies generated against 3′AMNic vaccine were evaluated usinga 3′AMNic-pGlu ELISA as follows: Dynatech Immulon 4 microtiter plates(Chantilly, Va.) were coated 100 μL/well with 10 ng/mL 3′AMNic-pGlu in0.1 M bicarbonate buffer, pH 9.6 and allowed to incubate overnight (ON)at room temperature (RT). The plates were then aspirated and blockedwith 1% BSA in PBS for one hour at RT. Samples and reference serum werediluted in PBB (1% BSA, 0.3% BRIJ in PBS, pH 7.2) to a dilution whichresults in an approximate optical density (OD) at 450 nm of 2.0. Theplates were washed (9% NaCl, 0.1% BRIJ) five times and diluted samplesand reference serum were loaded. The reference and samples were 2-folddiluted down the plates for a final volume of 100 μL/well and plates areincubated for 1 hour at 37° C. The plates were then washed again andloaded 100 μL/well with peroxidase-conjugated anti-species IgG, Fcspecific (Jackson, West Grove, Pa.) diluted in PBB and incubated at 37°C. for one hour. The plates were washed and incubated 10 minutes at RTwith 100 μl/well 3, 3′, 5, 5′-tetramethylbenzidine (TMB) substrate (KPL,Gaithersburg, Md.) diluted 1:1 with H₂O₂ (supplied with TMB reagentkit). The reaction is stopped with the addition of 100 μL/well 1 Mphosphoric acid and read at 450 nm on an MR4000 microtiter plate reader(Dynatech). Samples are quantified in relation to the reference usingparallel line analysis. The reference is assigned a numerical value(U/mL) that corresponds to the dilution which gives an OD ofapproximately 2.0 at 450 nm.

[0132] Antibody specificities were evaluated using an inhibition ELISAassay. Each anti-[3′ AMNic-Suc-rEPA] serum was diluted to aconcentration twice that which would results in an optical density ofabout 2.0 at 450 nm. Using the 3′ AMNic-pGlu ELISA described above, thediluted antiserum to be tested was absorbed 1:1 (v/v) with increasingamounts of test antigen (inhibitor) for three hours at 37° C., and thatabsorbed sample was tested in the ELISA using unabsorbed serum as areference. Percent absorption with reference to the unabsorbed samplewas determined for each sample.

[0133] The specificity of rat serum containing antibodies raised inresponse to 3′ AMNic-Suc-rEPA, using inhibition ELISA with nicotinetartrate as inhibitor, was calculated. The IC₅₀ for this antibody was3.5×10⁻⁶ M. The specificity of rabbit serum containing antibodies raisedin response to 3′ AMNic-Suc-rEPA, using inhibition ELISA with nicotinetartrate as inhibitor, was calculated. The IC₅₀ for this antibody was2.3×10⁻⁵ M.

EXAMPLE 11 Antibody Affinity and Binding Capacity

[0134] Antibody binding capacity was measured using equilibrium dialysisfor 4 hours at 37° C. using 0.7 mL of plasma, Teflon semi-micro cells,Spectrapor 2 membranes with a molecular weight cutoff of 12 to 14 kD andSorenson's buffer (0.13 phosphate, pH 7.4) see Pentel et al., J.Pharmacol. Exp. Ther. 246, 1061-1066 (1987). Plasma pH was measured atthe end of reach equilibrium dialysis run, and samples were used only ifthe final pH is 7.30 to 7.45.

[0135] Antibody affinity for nicotine was calculated using a solubleradioimmunoassay, see Mueller, Meth. Enzymol., 92, 589-601 (1983). Themolecular weight of IgG was measured tobe 150kD.

[0136] The binding constants and affinities obtained with theradioimmunoassay were as follows. For anti-[3′ AMNic-Suc-rEPA] ratserum, the IC₅₀ (Molar) was 1.36×10⁻⁷. The K_(a)(Molar-1) was 2.57×10⁷.Binding sites concentration was 2.61×10⁻⁶ binding sites/L andnicotine-specific IgG concentration was 0.2 mg/mL.

EXAMPLE 12 Evaluation of Nicotine Distribution in Plasma and Brain ofAnimal Models

[0137] The present inventive vaccine has been evaluated in variousanimal models. Rat models were used to determine the effect of active orpassive immunization on nicotine distribution in plasma and brain. Onestudy examined the effects of passive immunization on attenuation of thelocomotor effects of nicotine, which are a central nervous system (CNS)action of nicotine. Another experiment evaluated the effects of passiveimmunization on the effects of nicotine on the cardiovascular system:elevation of the systolic blood pressure.

[0138] To evaluate the present immunotherapy, an animal model has beendeveloped to simulate the rapid absorption of nicotine from twocigarettes in humans. This animal model is described in Hieda (1997) J.Pharmacol. Exp. Ther. 283(3):1076-1081. In this model, rats wereadministered 0.03 mg/kg of nicotine by i.v. infusion over 10 sec.,simulating the rapid absorption of nicotine from the lungs in humansmokers. Blood samples were taken at 3 and 10 min after nicotineinjection for measurement of plasma nicotine. When brain nicotineconcentrations were to be determined, animals were sacrificed 3 minafter nicotine injection, and their brains were quickly removed. Thevaccine of example 4 was evaluated in rats to determine its effect onthe distribution of nicotine in plasma and brain.

[0139] A. Active Immunization

[0140] Rats were immunized with the nicotine vaccine by three i.p.injections of 25 μg total per injection of vaccine (3′ AMNic-Suc-rEPA)two weeks apart. These animals had increased levels of nicotine inplasma 3 and 10 min after an infusion of 0.03 mg/kg of nicotine over 10seconds, compared with levels in non-immunized controls. See FIG. 1.Thus, active immunization was effective in increasing nicotine bindingin plasma. It is known that a modest reduction in the amount of nicotinereaching the brain can dramatically alter the behavioral effects ofnicotine.

[0141] B. Passive Immunization

[0142] With passive immunization, it was possible to determine the doseresponse effect of immune IgG in increasing plasma nicotine levels andreducing brain nicotine levels. Rats were administered with varyingamounts of anti-(3′ AMNic-Suc-rEPA) IgG (12.5 to 50 mg) total perinjection. As shown in FIG. 2, there was a clear dose responseeffect—increasing the dose of IgG increased serum nicotine and decreasedbrain nicotine levels.

[0143]FIG. 3 shows that anti-nicotine antibodies (anti-3′AMNic-Suc-rEPA) were present and active in the serum of rats, 30 min and1 day after administration of antibodies (50 mg) total per injection.FIG. 3 shows that following nicotine challenge (0.03 mg/kg infusion over10 seconds), these antibodies were effective in reducing nicotineconcentrations in brain and increasing nicotine levels in plasma, at 30minutes and 1 day after antibody administration.

[0144] Another demonstration of the efficacy of the passive immunizationwith nicotine vaccine of the invention is its ability to combatconsecutive infusions of nicotine. In a separate passive immunizationexperiments in rats, multiple doses of nicotine did not deplete theantibodies present or significantly reduce their capacity to bind tofreshly injected nicotine. In FIG. 4, 50 mg of anti-[3′ AMNic-Suc-rEPA]was infused at time zero. 24 hours later, five nicotine injections weremade -0.03 mg/kg nicotine was infused over 10 seconds, from the rightjugular vein, every 20 minutes for 80 minutes. A total of five nicotineinjections were made. The fifth injection of nicotine was spiked with³H-nicotine. Total blood and brain were collected 1 minute after thefifth injection. The results are shown below, and are graphicallyrepresented in FIGS. 4 and 5. Nicotine Concentrations (mean ± SD) Serum(ng/mL) Brain (ng/g) 1st dose 5th dose 5th dose Nicotine Nicotine3H-Nicotine Cotinine Nicotine 3H-Nicotine Immune IgG 245 ± 30 343 ± 46121 ± 17 30 ± 4 244 ± 33  90 ± 16 Control IgG 21 ± 3 55 ± 9 41 ± 4 35 ±5 257 ± 29 126 ± 14 % change +1067 +524 +195 −17 −13 −29

[0145] These results show that even after the fifth dose of nicotine,the antibodies are effective in increasing serum nicotine levels, anddecreasing brain nicotine levels. The results with the ³H-nicotinedemonstrate that antibodies are effective against the nicotine injectedat the fifth dose.

EXAMPLE 13 Evaluation of Locomotor Effects of Nicotine

[0146] This experiment used was designed to determine whether passiveimmunization could prevent an immediate CNS mediated action of nicotine.The rat model used in this experiment was developed by Dr. David Malinand is described in Malin et al. Nicotine—specific IgG reduceddistribution to brain and attenuates its behavioral and cardiovasculareffects in rats, submitted to the Fifth Annual Meeting of the Societyfor Research on Nicotine and Tobacco, San Diego, Calif., Mar. 5-7, 1999.To establish a baseline, the effect of a subcutaneous injection of 0.8mg/kg of nicotine tartrate on locomotor activity level of rats wasmeasured. The 0.8 mg/kg dose of nicotine tartrate is the highest dosethat could be used without inducing locomotor abnormalities.

[0147] There was an increase in activity level after nicotine injectionin rats that were not pre-treated with anti-[3′ AMNic-Suc-rEPA], and inrats that were pretreated with 50 mg of normal rabbit serum IgG. SeeFIG. 6A, right bar and FIG. 6B, left bar. This effect was suppressed bypretreating the animals with 50 mg of anti-[3′ AMNic-Suc-rEPA] immuneIgG (FIG. 6B, right bar). This shows that the anti-nicotine antiserumeliminated a stimulant effect of nicotine, in vivo.

EXAMPLE 14 Evaluation of Nicotine on Systolic Blood Pressure

[0148] In this experiment, another indicator of the behavioral effect ofnicotine was measured: the change in systolic blood pressure. Rats werepretreated with anti-[3′ AMNic-Suc-rEPA] IgG, or control IgG. Rats weretreated with a subcutaneous injection of 0.1 mg/kg nicotine tartrate.Control rats showed an increase in systolic blood pressure of 42.6±3.2mm Hg, when treated with nicotine. When rats were pretreated withanti-nicotine antiserum IgG, the nicotine challenge was less effective.When increasing amounts of anti-nicotine serum were administered, thisdiminished the ability of nicotine to raise blood pressure. As shown inFIG. 7, there was a negative linear trend of blood pressure as afunction of IgG dose.

What is claimed is:
 1. A hapten-carrier conjugate of the followingformula:

wherein m is 1 to 2500, n is 0 to 12, y is 1 to 12, X is selected fromthe group consisting of NH—CO, CO—NH, CO—NH—NH, NH—NH—CO, NH—CO—NH,CO—NH—NH—CO, and S—S; Y is selected from the group consisting of NH—CO,CO—NH, CO—NH—NH, NH—NH—CO, NH—CO—NH, CO—NH—NH—CO, and S—S, and the—(CH₂)_(n)—X—(CH₂)_(y)—Y— moiety is bonded to the 3′, 4′ or 5′ position.2. The conjugate of claim 1, wherein m is 11 to 17, n is 1, y is 2, X isNH—CO, Y is CO—NH, wherein the carrier protein is exoprotein A and the—(CH₂)_(n)—X—(CH₂)_(y)—Y— is bonded to the 3′ position.
 3. The conjugateof claim 1, wherein m is 11 to 17, n is 1, y is 2, X is NH—CO, Y isCO—NH, wherein the carrier protein is exoprotein A and the—(CH₂)_(n)—X—(CH₂)_(y)—Y— is bonded to the 4′ position.
 4. The conjugateof claim 1, wherein m is 11 to 17, n is 1, y is 2, X is NH—CO, Y isCO—NH, wherein the carrier protein is exoprotein A and the—(CH₂)_(n)—X—(CH₂)_(y)—Y— is bonded to the 5′ position.
 5. The conjugateof claim 1, wherein m is selected from the group consisting of 1 to 20and 1 to
 200. 6. A hapten-carrier conjugate of the following formula:

wherein n is 0 to 12, j is 1 to 1000, k is 1 to 20, and E is an aminoacid-containing matrix.
 7. The hapten-carrier conjugate of claim 6,wherein the matrix is poly-L-glutamic acid.
 8. An antibody produced inresponse to the hapten-carrier conjugate of claim
 1. 9. A functionalfragment of the antibody of claim
 8. 10. The antibody of claim 8, whichis a monoclonal antibody.
 11. The antibody of claim 8, which is apolyclonal antibody.
 12. An antibody produced in response to thehapten-carrier conjugate of claim
 6. 13. A functional fragment of theantibody of claim
 12. 14. The antibody of claim 12, which is amonoclonal antibody.
 15. The antibody of claim 12, which is a polyclonalantibody.
 16. A method of treating nicotine addiction in a patient inneed of such treatment comprising administering a therapeuticallyeffective amount of the hapten-carrier conjugate of claim
 1. 17. Amethod of treating nicotine addiction in a patient in need of suchtreatment comprising administering a therapeutically effective amount ofthe antibody of claim
 8. 18. A method of preventing nicotine addictionin a patient in need of such prevention, comprising administering atherapeutically effective amount of the hapten-carrier conjugate ofclaim
 1. 19. A method of preventing nicotine addiction in a patient inneed of such treatment comprising administering a therapeuticallyeffective amount of the antibody of claim
 8. 20. The method of claim 16,further comprising administering a compound that is useful in thetreatment of addiction.
 21. A method of treating nicotine addiction in apatient in need of such treatment comprising administering atherapeutically effective amount of the hapten-carrier conjugate ofclaim
 6. 22. The method of claim 21, further comprising administering acompound that is useful in the treatment of addiction.
 23. A method oftreating nicotine addiction in a patient in need of such treatmentcomprising administering a therapeutically effective amount of theantibody of claim
 12. 24. A method of preventing nicotine addiction in apatient in need of such prevention, comprising administering atherapeutically effective amount of the hapten-carrier conjugate ofclaim
 6. 25. A method of preventing nicotine addiction in a patient inneed of such treatment comprising administering a therapeuticallyeffective amount of the antibody of claim
 12. 26. A process forproducing an antibody, comprising immunizing a host mammal with thehapten-carrier conjugate of claim
 1. 27. The process of claim 26,wherein the antibody is monoclonal.
 28. The process of claim 26, whereinthe antibody is polyclonal.
 29. A process for producing an antibody,comprising immunizing a host mammal with the hapten-carrier conjugate ofclaim
 6. 30. The process of claim 29, wherein the antibody ismonoclonal.
 31. The process of claim 29, wherein the antibody ispolyclonal.
 32. A vaccine composition comprising at least one conjugateof claim
 1. 33. A vaccine composition comprising at least one conjugateof claim
 6. 34. A kit for determining the presence of nicotine in asample, comprising an antibody of claim
 8. 35. A kit for determining thepresence of nicotine in a sample, comprising an antibody of claim 12.